Liquid chromatography (LC) is a popular technique for performing an analytical separation of a sample into constituent components, i.e., the analytes of interest to a researcher. As readily known by persons skilled in the art, during the course of a chromatographic separation, the sample is transported in a mobile phase, which is a liquid in LC techniques. The mobile phase is forced through a stationary phase that is immiscible relative to the mobile phase. Typically, the stationary phase is supported in a column or cartridge through which the sample and mobile phase flow. The respective compositions of the mobile phase and stationary phase are selected to cause the analytes of the sample to become distributed between the mobile phase and stationary phase to varying degrees dependent on the nature of the respective analytes. Analytes that are strongly retained by the stationary phase travel slowly with the mobile phase, while analytes that are weakly retained by the stationary phase travel more rapidly. As a result, analytes of differing compositions become separated from each other as the mobile phase flows through the column. In this manner, the analytes are in effect sorted sequentially as the eluent flows out from the column, thereby facilitating their analysis by a suitable analytical instrument.
One particularly useful instrument for analyzing the separated components of the sample is a mass spectrometer. Several different types of mass spectrometers are commercially available and well known to persons skilled in the art. The advent of mass spectrometers that utilize an atmospheric pressure ionization (API) interface between the sample input and the input into the mass analyzing and detection portions of the mass spectrometer, such as an electrospray ionization (ESI) source or an atmospheric pressure chemical ionization (APCI) source, has enabled the eluent from an LC column to be fluidly connected to the mass spectrometer. The resulting system is commonly termed a liquid chromatography/mass spectrometry (LC/MS) system.
Generally, the API interface of a mass spectrometer converts the column eluent, i.e., an analyte/mobile phase matrix, into droplets that must be vaporized or desolvated so that the analytes can be ionized in preparation for processing in the evacuated regions of the mass spectrometer. Typically, a stream of an inert drying gas such as diatomic nitrogen is flowed into the API interface to enhance evaporation of the droplets. Often, a separate stream of nebulizing gas such as nitrogen is also flowed into the API interface to assist in nebulizing the analyte/mobile phase matrix. The drying gas, and also the nebulizing gas, can be heated prior to introduction into the API interface. Conventionally, both of these API gases are flowed under constant temperature and flow rate conditions.
In the operation of an LC/MS system, it is important to optimize both the efficiency of the separation accomplished by the LC column so that differing analytes are properly separated within a reasonable run time, and the efficiency of the mass spectrometer so that the mass spectrum produced thereby maximizes the signal-to-noise ratio (S/N) and the resolution of the chromatographic peaks. The process of vaporizing the analyte/mobile phase matrix in the API interface can affect the performance of the mass spectrometer and hence the analytical value of the mass spectrum produced thereby.
Traditionally, a chromatographic separation has entailed isocratic elution in which the mobile phase consists of a single solvent of constant composition. For the afore-mentioned API interface operating according to fixed temperature and flow of the API gases, optimal efficiency of droplet evaporation is only achieved for a single composition of liquid chromatographic solvents. For isocratic separations, these fixed conditions are acceptable in most cases.
It has become increasingly desirable, however, to perform a chromatographic separation that entails gradient elution. In a gradient elution, the mobile phase consists of a multi-solvent system (typically two or three solvents differing significantly in polarity and volatility), and the ratio of the respective solvents in the mobile phase composition is varied continuously or step-wise over time in a programmed or at least predetermined manner. For example, in reversed-phase liquid chromatography, the gradient profile can entail steadily increasing the percentage of an organic solvent in the mobile phase while decreasing that of a less volatile solvent such as water. Gradient elution can significantly improve separation efficiency, and can be employed to change the retention factor of the mobile phase to improve chromatographic resolution of two or more species.
Unfortunately, when gradient elution is performed, the fixed conditions for the API gases result in less than optimal efficiency in the API interface as the mobile phase conditions deviate from the essentially single solvent composition that was used to set up the analysis. As the composition of the mobile phase changes according to the gradient profile, properties such as the surface tension of the droplets, the viscosity of the mobile phase, and the volatility of the mobile phase likewise change. It is believed that prior to the subject matter disclosed herein, there has been no means to adequately compensate for the effects of varying the composition of the mobile phase, particularly at one or the other extreme region of the gradient. As a consequence, the evaporation of droplets and the signals detected by the mass spectrometer have not been optimized for all analytes across the gradient separation. Moreover, there has been an unacceptable opportunity for thermal degradation of a given analyte to occur under gas conditions that are too aggressive for a mobile phase composition associated with that analyte at a particular instance.
In view of the foregoing, it would be advantageous to operate an LC/MS system set up for gradient elution in a manner that optimizes evaporation of the droplets in the API interface in response to the varying mobile phase composition, thereby improving signal detection and reducing the risk of thermal degradation. The subject matter disclosed herein addresses, in whole or in part, these and other problems associated with analytical techniques involving gradient elution.